The overall goal of this proposal is to develop gene-based therapies for inherited disorders of skin. Cutaneous gene therapy research continues to focus on developing efficient gene transfer methods that lead to long-term transgene expression. Considerable success has been achieved with improvements in vectors and in devising strategies for targeting stem cells. However, we lack an understanding of host responses when a neoantigen is expressed in skin. These responses are likely to complicate cutaneous gene therapy trials, especially when a patient has a null mutation. Our studies demonstrate that when a neoantigen is expressed in the skin of normal mice, transgene expression is curtailed and the transduced cells are rejected through immune responses. The knowledge gained from related studies in liver and muscle is not likely to be applicable to skin as, unlike those tissues, skin has immune-associated functions. The aim of this research proposal therefore, is to develop an improved understanding of skin-specific immune responses to neoantigen and to develop strategies for avoiding these responses. Based on an emerging body of knowledge we hypothesize that host immune responses are most amenable to modulation at the point at which antigens expressed in keratinocytes are cross-presented to T cells. The experiments described in this proposal are designed to delineate the mechanism of antigenic transgene presentation and priming of T cells and to explore strategies to divert these responses toward less destructive or tolerogenic ones. Three specific aims are proposed: 1) To examine the role of cross-presentation of keratinocyte-derived antigens in inducing T cell immunity; 2) To delineate the role of inflammation in priming of T cells by antigen presenting cells; and 3) To induce a state of tolerance to keratinocyte-derived neoantigens by modulating the signaling events involved in initiation of T cell immunity. It is expected that development of new strategies to circumvent destructive immune responses will overcome a major obstacle to clinical applications of skin gene therapy.